Mycobacterium tuberculosis strain with deletions in menT3 and menT4 is attenuated and confers protection in mice and guinea pigs

The genome of Mycobacterium tuberculosis encodes for a large repertoire of toxin-antitoxin systems. In the present study, MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems have been functionally characterized. We demonstrate that ectopic expression of these toxins inhibits bacterial growth and this is rescued upon co-expression of their cognate antitoxins. Here, we show that simultaneous deletion of menT3 and menT4 results in enhanced susceptibility of M. tuberculosis upon exposure to oxidative stress and attenuated growth in guinea pigs and mice. We observed reduced expression of transcripts encoding for proteins that are essential or required for intracellular growth in mid-log phase cultures of ΔmenT4ΔT3 compared to parental strain. Further, the transcript levels of proteins involved in efficient bacterial clearance were increased in lung tissues of ΔmenT4ΔT3 infected mice relative to parental strain infected mice. We show that immunization of mice and guinea pigs with ΔmenT4ΔT3 confers significant protection against M. tuberculosis infection. Remarkably, immunization of mice with ΔmenT4ΔT3 results in increased antigen-specific TH1 bias and activated memory T cell response. We conclude that MenT3 and MenT4 are important for M. tuberculosis pathogenicity and strains lacking menT3 and menT4 have the potential to be explored further as vaccine candidates.


Ectopic expression of MenT3 and MenT4 results in growth inhibition
In order to investigate the effect of overexpression of MenT3 or MenT4 on the growth of E. coli or M. tuberculosis, wild type toxins or their mutants were individually cloned in either pET28b (IPTG inducible) or pTetR (Atc inducible) expression vectors.As reported earlier, overexpression of MenT3 or MenT4 inhibited E. coli growth in comparison to uninduced cultures (Fig. 1a, b) 30 .Further, G62, K189, D208 in motifs I, III and IV of MenT3 and G51, D71 in motifs I and II of MenT4 were mutated, cloned in pET28b and growth assays were performed (Fig. S1a).As expected, overexpression of either MenT3 G62A or MenT3 K189A or MenT3 D208A or MenT4 G51A or MenT4 D71A proteins did not inhibit E. coli growth (Figs.1a, b and S1b).Next, to verify whether the co-expression of antitoxins can alleviate the growth inhibition activity of MenT3 and MenT4 toxins, we cloned them along with their cognate or non-cognate antitoxin in MCS-I and MCS-II of an IPTG inducible expression system, pETDuet.We observed that growth inhibition associated with MenT3 and MenT4 overexpression was restored by coexpression of their cognate antitoxins, MenA3 and MenA4, respectively (Fig. 1c, d).However, no growth restoration was seen upon coexpression of MenT3 and MenT4 with their non-cognate antitoxins (Fig. 1c, d).In agreement with E. coli data, we observed that inducible expression of either MenT3 or MenT4 also inhibited M. tuberculosis growth (Fig. 1e).In comparison to strain harboring vector control, overexpression of MenT3 and MenT4 reduced the bacterial growth by ~5.0-fold at 4 days post-induction (Fig. 1f).This growth defect upon overexpression of either MenT3 or MenT4 increased to ~28.0-and 15.0-fold, respectively, at 7 days post-induction (Fig. 1f).

Deletion of both menT3 and menT4 increases the susceptibility of M. tuberculosis to oxidative stress
The abundance of TA systems in members of the M. tuberculosis complex raises a possibility that these might function in a cumulative manner and contribute to M. tuberculosis survival in stress conditions and host tissues 11,12,14 .In order to understand the role of MenT3 and MenT4 in the physiology of M. tuberculosis, we constructed ΔmenT3, ΔmenT4 single mutant and ΔmenT4ΔT3 double mutant strain using temperature-sensitive mycobacteriophages (Fig. 1g).As shown in Fig. 1h, PCR amplification using menT3 locus-specific primers resulted in amplifications of sizes 882 bp, 1.5 kb and 1.5 kb using genomic DNA isolated from wild type, ΔmenT3 and ΔmenT4ΔT3 strains of M. tuberculosis, respectively.PCR amplification of sizes 885 bp, 2.0 kb and 2.0 kb were obtained from genomic DNA isolated from wild type, ΔmenT4 and ΔmenT4ΔT3 strains of M. tuberculosis, respectively, using menT4 locus primers (Fig. 1h).Whole genome sequencing revealed that sequences aligning to menT3 and menT4 locus were missing from the reads obtained from ΔmenT4ΔT3 genomic DNA in comparison to reads obtained from the wild type strain (Fig. S2).These observations confirmed that the open reading frame for menT3 and menT4 has been replaced with kanamycin and hygromycin resistance gene, respectively, in the ΔmenT4ΔT3 strain of M. tuberculosis.
We next compared the growth patterns of wild type and ΔmenT4ΔT3 M. tuberculosis strains in liquid culture.We observed that both strains displayed comparable bacterial counts during various stages of growth in vitro.We also compared the survival of parental and ΔmenT4ΔT3 strains after exposure to various stress conditions.As shown in Fig. 2a, relative to the parental strain, ΔmenT4ΔT3 strain was ~8.5-fold more susceptible to oxidative stress after exposure for 24 h.However, at 72 h post-exposure to oxidative stress, ΔmenT4ΔT3 showed ~225.0-foldincreased susceptibility in comparison to the parental strain (Fig. 2a).We also observed that complementation of the double mutant strain with menT3 partially restored this growth defect (Fig. S3a).However, we were unable to restore this growth defect of double mutant strain upon complementation with menT4 (Fig. S3a).qPCR studies revealed that the transcript levels for menT3 and menT4 were restored in their respective single-complemented strain (Fig. S5a).We also found that the sensitivity of wild type, ΔmenT3 and ΔmenT4 was comparable after being exposed to oxidative stress (Fig. S3c, d).Previously, it has been demonstrated that a subset of toxins is induced in response to stress conditions such as low oxygen, nutrient limiting, macrophage engulfment or drug exposure.The increased transcription and synthesis of toxins lead to TA systems activation and subsequent growth inhibition 12,21,22,32 .Since ΔmenT4ΔT3 was susceptible upon exposure to oxidative stress relative to the parental strain, we next measured the relative levels of menT3 and menT4 in these conditions.We observed that the transcript levels of menT3 and menT4 remained unchanged after being exposed to oxidative stress (Fig. S3b).The survival of parental, ΔmenT3, ΔmenT4 and ΔmenT4ΔT3 strains was comparable upon exposure to either nitrosative or nutritional or acidic stress (Figs.2b-d and S3e-j).We also compared the ability of wild type, ΔmenT3, ΔmenT4 and ΔmenT4ΔT3 to infect and survive inside THP-1 macrophages.The growth patterns of these strains were similar at days 2, 4 and 6 post-infection in macrophages (Figs.2e and S4a, b).Taken together, these observations suggest that MenT3 and MenT4 are mutually redundant for in vitro growth, and simultaneous deletion of both menT3 and menT4 increased the susceptibility of M. tuberculosis to oxidative stress.

Deletion of both menT3 and menT4 impairs the virulence of M. tuberculosis in guinea pigs
Previously, we have reported that simultaneous deletion of MazF ribonucleases mazF3, mazF6 and mazF9 or higB1 or vapBC3 or vapBC4 or vapBC11 or vapC22 significantly reduced M. tuberculosis growth in guinea pigs 21,23,[25][26][27] .However, deletions in either relE1 or relE2 or relE3 or vapC28 or vapC21 or darTG did not reduce M. tuberculosis growth in guinea pigs or mice 21,24,33,34 .In addition to type II TA systems, we have recently shown that MenT2 toxin belonging to the MenAT subfamily is also essential for M. tuberculosis pathogenesis in guinea pigs 35 .Previously, high throughput screening assays such as transposon site hybridization (TRASH) and designer array for defined mutant analysis (DeADMAn) have been performed to identify genes necessary for in vivo growth of M. tuberculosis 36,37 .According to these studies, menT3 and menT4 are mutually redundant and not required for M. tuberculosis to establish infection in mice 36,37 .In the present study, we compared the growth of wild type and ΔmenT4ΔT3 strains in aerosolinfected guinea pigs.We observed that lung bacillary loads were decreased by ~6.6-and 950.0-fold in guinea pigs infected with ΔmenT4ΔT3 at 4-and 8 weeks post-infection, respectively, relative to parental strain infected guinea pigs (Fig. 2f).As shown in Fig. 2g, we observed ~117.0-and~3750.0-foldreduction in bacterial loads in spleens of ΔmenT4ΔT3 infected guinea pigs relative to wild type strain infected guinea pigs at 4-and 8-weeks post-infection, respectively.In agreement with bacterial burdens, H&E-stained lung sections from guinea pigs infected with wild type strain displayed increased cellular infiltration and severely reduced alveolar spaces (Fig. 2i).This increased cellular infiltration indicates severe inflammation and pathology in lung tissue sections of wild type strain infected guinea pigs at both time points (Fig. 2i).In comparison, the histologically stained lung sections of ΔmenT4ΔT3 infected guinea pigs demonstrated intact lung architecture and large alveolar spaces at both time points (Fig. 2i).At both time points, the total granuloma score in lungs of ΔmenT4ΔT3 infected guinea pigs was significantly decreased by ~10.0-fold in comparison to total granuloma score in lung sections from guinea pigs infected with the wild type strain (Fig. 2h).Taken together, we demonstrate that simultaneous deletion of menT3 and menT4 results in attenuation of M. tuberculosis growth in guinea pigs.

Deletion of both menT3 and menT4 alters the transcriptional profiles of M. tuberculosis
We next compared the transcriptional profiles of mid-log phase cultures of wild type and ΔmenT4ΔT3 strain to identify the differentially expressed pathways following the simultaneous deletion of menT3 and menT4 in M. tuberculosis.Using a 2.0-fold cut-off and p adj value of ≤0.05, we observed that compared to the parental strain, transcripts encoding for 36 and 51 proteins were increased and decreased, respectively, in ΔmenT4ΔT3 (Supplementary Data 1).The DEGs were further annotated as per the functional category listed by mycobrowser (https://mycobrowser.epfl.ch/).Approximately, 33 and 23% of the transcripts with differential expression encoded for either conserved hypothetical proteins or proteins involved in intermediary metabolism and respiration (Table 1).Interestingly, we observed that the expression of toxins belonging to type II TA systems such as Erd-man_0269 (Rv0240, vapC24), Erdman_0658 (Rv0598c, vapC27), Erd-man_2181 (Rv1982c, vapC36) and Erdman_2744 (Rv2494, vapC38) was increased in mid-log phase cultures of ΔmenT4ΔT3 relative to the wild type strain (Supplementary Data 1).We also noticed that relative to the parental strain, the levels of transcripts encoding for proteins involved in M. tuberculosis adaptation in host tissues were significantly reduced in the ΔmenT4ΔT3.These included Erdman_0097 (Rv0081), a regulatory protein known to be upregulated upon exposure to low oxygen and Erdman_0100 (Rv0084, hycD), a protein involved in formate metabolism 38,39 .In addition to these, the transcript levels of Erd-man_0321 (Rv0287, esxG), a protein involved in the inhibition of phagosome maturation and pathogenesis of M. tuberculosis, were also reduced in the ΔmenT4ΔT3 strain (Supplementary Data 1) 40 .As shown in Supplementary Data 1, the levels of transcripts encoding for proteins essential for growth (Erdman_3437 (Rv3137), Erdman_3739 (Rv3418c), stress adaptation (Erdman_0630 (Rv0574c), Erdman_2884 (Rv2624c)) and interaction with host Interferon-γ (IFN-γ) (Erdman_1325, Rv1183) were also reduced in ΔmenT4ΔT3 strain relative to the parental strain 36,[41][42][43][44] .Several studies have reported that replacing an open reading frame with an antibiotic selection marker might affect the expression of neighboring genes due to the polar effect.However, we observed that the relative levels of menT3 and menT4 neighboring genes were comparable in both wild type and ΔmenT4ΔT3 strains (Fig. S5b and Supplementary Data 1).Another possible explanation for the observed attenuated phenotype of ΔmenT4ΔT3 in vivo might be the loss of apolar lipids such as PDIMs during in vitro culturing 45 .In order to rule out this possibility, we compared polar and apolar lipid profiles of mid-log phase cultures of wild type, ΔmenT3, ΔmenT4 and ΔmenT4ΔT3 strains.We observed that the relative levels of apolar lipids (PDIMs, TAG), mycolic acids (MAMEs, FAMEs) and polar lipids (TAG, FAM and DAG) were comparable in wild type and various mutant strains (Fig. S5c).Overall, the data suggests that the attenuated phenotype of the ΔmenT4ΔT3 strain in guinea pigs is most likely associated with reduced levels of transcripts encoding for proteins required for either stress adaptation or virulence of M. tuberculosis.

Deletion of both menT3 and menT4 results in attenuation of M. tuberculosis growth in mice
We next compared the growth of wild type and ΔmenT4ΔT3 strain in aerosol-infected Balb/c mice at 4 and 8-weeks post-infection.As shown in Fig. 3a, b, the lung and splenic bacillary loads in ΔmenT4ΔT3 infected mice were decreased by 10.0-and 14.0-fold, respectively, in comparison to wild type infected mice at 4-weeks post-infection.However, at 8 weeks post-infection, we observed 7.0-and 13.0-fold reduction in lungs and splenic bacillary loads in ΔmenT4ΔT3 infected mice compared to mice infected with the wild type strain (Fig. 3a, b).As shown in Fig. S4c, d, we were unable to restore this growth defect in the lungs and spleens of infected animals upon complementation of the double mutant strain with either menT3 or menT4.These observations suggest that both MenT3 and MenT4 contribute cumulatively to the ability of M. tuberculosis to establish infection in host tissues.We next performed RNA-seq analysis of lung tissues of uninfected mice and those infected with either parental or ΔmenT4ΔT3 strains at 4-weeks postinfection to understand the plausible underlying mechanisms associated with the attenuated phenotype of the double mutant strain in vivo.Using a p adj value of ≤ 0.05 and a cut-off fold change value of 4.0 and −4.0, we observed that relative to uninfected animals, the expression of 826 and 423 transcripts were increased or decreased in mice infected with the parental strain (Figs.3c, S4e and Supplementary Data 2).As shown in Fig. 3c, the transcript levels of 312 and 119 genes were increased or decreased, respectively, in mice infected with the ΔmenT4ΔT3 strain relative to uninfected animals (Fig. S4f and Supplementary Data 3).The transcript levels of 330 and 160 genes were increased or decreased in animals infected with ΔmenT4ΔT3 strain in comparison to profiles obtained from parental strain-infected mice (Fig. 3c, d and Supplementary Data 4).
We noticed that relative to the parental strain, the transcript levels of genes encoding for proteins involved in calcium signaling were increased in animals infected with ΔmenT4ΔT3 strain (Fig. 3e and Supplementary Data 4).Previously, it has been shown that increased calcium levels are associated with the induction of numerous antimicrobial pathways such as autophagy and apoptosis 46 .Previously, it has been reported that Adcy1 is activated by apoA-1 to promote cholesterol efflux from THP-1 macrophage foam cells 47 .The formation of foamy macrophages is associated with disease progression that leads to cavitation and release of infectious bacilli 48 .In agreement, we observed ~4.0-fold increased expression of adcy1 in the lungs of mice infected with ΔmenT4ΔT3 strain relative to the parental strain (Supplementary Data 4).M. tuberculosis inhibits phagolysosome fusion by interfering with phosphatidylinositol 3-phosphate (PI3P) signaling 49 .We observed increased expression of adra1a in lung tissues of ΔmenT4ΔT3 strain-infected mice compared to the parental strain (Supplementary Data 4).Adra1a is involved in the release of inositol 1,4,5-triphosphate and the activation of protein kinase C [50][51][52] .Studies have shown that deletion of protein kinase C results in higher susceptibility to TB due to increased lung pathology, the release of proinflammatory cytokines and bacterial burdens 53,54 .Numerous studies have shown that bacterial pathogens are able to establish persistent infection by subverting autophagy 55 .We observed that the levels of transcripts encoding for proteins involved in apoptosis and autophagy, such as slc4a1, bub1, nod1, rnf152, atg16l1, gba, tsc1, phf23, map2k7, mfn2 and wnt11, were differentially expressed in lung tissues of mice infected with ΔmenT4ΔT3 strain compared to the parental strain (Fig. 3f and Supplementary Data 4).Autophagy acts as an immune effector mechanism, resulting in phagosomal maturation that mediates mycobacteria clearance 56 .Studies have shown that Bub1 and Phf23 act as negative regulators of autophagy [57][58][59] .As shown in Supplementary Data 4, we observed reduced expression of bub1 and phf23 transcript in lung tissues of ΔmenT4ΔT3 strain infected mice relative to the parental strain infected animals.Additionally, we observed that the levels of transcripts encoding for atg16l1 were increased in ΔmenT4ΔT3 infected mice lung tissues (Fig. 3f and Supplementary Data 4).Previously it has been shown that depletion of Atg16l1 is associated with increased M. tuberculosis growth and susceptibility in mice 60 .The reduced levels of ptpro also suggest reduced inflammation and enhanced apoptosis in lung tissues of ΔmenT4ΔT3 infected animals relative to mice infected with the parental strain 61 (Supplementary Data 4).We also observed an increased level of wnt11 transcript in lung tissues of ΔmenT4ΔT3 strain infected mice compared to the parental strain infected animals (Supplementary Data 4).Previously, it has been shown that overexpression of wnt11 in intestinal epithelial cells decreased Salmonella invasion and inhibited bacteria induced intestinal inflammation 62 .Furthermore, we observed varied expression of several transcripts associated with immune response in ΔmenT4ΔT3 strain infected mice relative to the parental strain infected mice (Supplementary Data 4).We observed reduced levels of transcripts encoding for various cytokines and chemokines such as il-21, ccl1, cxcl5, cxcl3, ccl4, and ccl21a in ΔmenT4ΔT3 infected mice (Fig. 3g and Supplementary Data 4).Studies have shown that increased expression of Ccl1 is associated with increased endoplasmic reticulum stress and

7.
Cell wall and cell processes 10 6

9.
Information pathways 1 3 10. Stable RNA -1 The number of upregulated and downregulated genes in the ΔmenT4ΔT3 strain classified as per their functional annotation are shown.The data were obtained from two biological replicates.
granuloma formation during mycobacterial treatment 63 .Further, it has been reported that elevated plasma levels of Ccl1, Ccl3, Cxcl1, Cxcl9, and Cxcl10 correlate with disease severity in infected individuals 64 .Furthermore, Cxcl5 secreted by pulmonary epithelial cells contributes to excessive neutrophilic inflammation, and mice deficient in Cxcl5 exhibit enhanced survival upon high-dose of M. tuberculosis infection compared to wild type mice 65 .The transcript levels of sag, hamp, il1rl1, lck, and pla2g5 that encode for proteins involved in the activation of macrophages, neutrophils or T-cells were also increased in lung tissues of mice infected with the mutant strain in comparison to lung tissues from parental strain infected mice (Fig. 3g and Supplementary Data 4) [66][67][68][69] .It has also been reported that increased levels of Pla2g5 result in enhanced adaptive immune response and phagocytosis of bacteria by macrophages 70,71 .Taken together, these observations suggest that the in vivo attenuated phenotype of ΔmenT4ΔT3 in lung tissues is most likely associated with increased expression of proteins involved in either calcium homeostasis, apoptosis or autophagy along with decreased expression of transcripts associated with inflammatory response.
Immunization with ΔmenT4ΔT3 strain imparts protection in mice and guinea pigs against M. tuberculosis challenge Studies have shown that immunization of animals with live attenuated M. tuberculosis strains provides long-term protection against challenge with virulent strain as these strains closely mimic the antigenic repertoire of the infectious agent [72][73][74] .Since M. tuberculosis is an intracellular pathogen with pulmonary pathology driven by IFNγ response, C57BL/6 is a widely used strain to study immunological responses, DC-NK crosstalk and T H 1 cellular responses [75][76][77][78][79][80] .Since ΔmenT4ΔT3 was significantly attenuated for growth in guinea pigs, we next evaluated whether immunization with this strain imparts protection against M. tuberculosis challenge in C57BL/6 mice (Fig. 4a).We found that the number of immunizing bacilli (Mycobacterium bovis Bacille Calmette-Guerin Pasteur, BCG and ΔmenT4ΔT3) in lungs and spleens of immunized C57BL/6 mice were below the detection limit at 6 weeks postimmunization.In comparison to naive mice, vaccination with ΔmenT4ΔT3 reduced lung and splenic loads of M. tuberculosis by ~7.5and 5.0-folds, respectively, at 4 weeks post-infection (Fig. 4b, c).In comparison to naive mice, immunization with BCG reduced the bacterial counts by 32.0-fold and 7.5-fold in lungs and spleens, respectively, at 4 weeks post-infection (Fig. 4b, c).At 10 weeks post-challenge with M. tuberculosis, immunization with ΔmenT4ΔT3 reduced the bacterial numbers by ~4.5-fold and 18.5-fold in lungs and spleens, respectively, in comparison to naive mice (Fig. 4d, e).At 10 weeks postinfection, in comparison to naive mice, immunization with BCG reduced lungs and splenic bacillary loads by ~8.5-fold and 4.4-fold, respectively (Fig. 4d, e).We observed that immunization with ΔmenT4ΔT3 imparted ~4.0-fold increased protection in comparison to immunization with BCG in spleens at 10 weeks post-infection (Fig. 4e).Next, we assessed the ability of ΔmenT4ΔT3 to impart protection against challenge with M. tuberculosis in guinea pigs (Duncan Hartley strain) (Fig. 4f).As observed in C57BL/6 mice, the lung and splenic bacillary loads in BCG and ΔmenT4ΔT3 immunized guinea pigs were below the detection limit at 6 weeks post-immunization.In comparison to naive guinea pigs, we observed ~70.0-fold and 6.0-fold reduction in bacterial numbers in the lungs of ΔmenT4ΔT3 and BCG vaccinated guinea pigs, respectively, at 4 weeks post-infection (Fig. 4g).We observed ~11.0-fold reduction in lung bacillary loads in ΔmenT4ΔT3 immunized animals in comparison to BCG vaccinated guinea pigs (Fig. 4g).Further, we observed immunization with either BCG or ΔmenT4ΔT3 reduced lung bacillary loads by ~11.0-and 27.0fold, respectively, in comparison to naive animals at 8 weeks postchallenge (Fig. 4h).However, ~2.5-fold increased protection in ΔmenT4ΔT3 immunized animals in comparison to BCG immunized guinea pigs was not statistically significant (Fig. 4h).We also performed histopathology analysis of lung sections to determine the extent of disease progression in unvaccinated and vaccinated guinea pigs at both time points.As shown in Fig. 4i, j, the total granuloma score in lung sections of naive guinea pigs was ~50.0 at both time points.In comparison, the total granuloma score in guinea pigs immunized with BCG was reduced by 1.70-fold in comparison to naive animals at 8-weeks post-infection (Fig. 4j).In comparison to naive guinea pigs, immunization with ΔmenT4ΔT3 significantly reduced the total granuloma score by 5.5-and 124.0-fold at 4-and 8-weeks postinfection, respectively (Fig. 4i, j).The total granuloma score was reduced by 4.5-and 73.0-fold in ΔmenT4ΔT3 immunized guinea pigs in comparison to guinea pigs immunized with BCG at 4-and 8-weeks post-infection, respectively (Fig. 4i, j).The histopathological analysis revealed large granulomas and significant tissue damage in H&Estained lung sections from naive animals.In comparison, the number of tubercles and tissue damage was reduced in guinea pigs immunized with BCG (Fig. 4k).In agreement with the total granuloma score, we observed normal lung architecture and few granulomas in the H&Estained section of guinea pigs immunized with ΔmenT4ΔT3 at both time points (Fig. 4k).Taken together, these observations suggest that immunization with ΔmenT4ΔT3 resulted in significant protection against challenge with M. tuberculosis in both mice and guinea pigs.

Immunization of mice with ΔmenT4ΔT3 induces a T H 1-biased response and activated memory T cell response
In order to determine immune correlates of protection in ΔmenT4ΔT3 immunized mice, we evaluated the antigen-specific adaptive T cell response in spleens of naive, BCG and ΔmenT4ΔT3 vaccinated C57BL/6 mice at 6 weeks post-immunization (Fig. S6).We observed that immunization of mice with ΔmenT4ΔT3 promoted antigen-specific T H 1 response by increasing the frequency of IFN-γ + CD4 + T-cells by ~270% and 190% in comparison to naive and BCG immunized mice, respectively (Fig. 5a, b).Also, in comparison to naive mice, immunization with BCG resulted in ~145% increase in the frequency of IFN-γ + CD4 + T-cells (Fig. 5a, b).Similarly, we observed an increase in the frequency of IFNγ + CD8 + T-cells by ~300-450% in ΔmenT4ΔT3 vaccinated mice compared to naive and BCG immunized mice (Fig. 5d, e).T-bet is the master transcription factor for T H 1 cells and also negatively regulates T H 2 immune response 81 .In comparison to naive mice, immunization with ΔmenT4ΔT3 significantly enhanced the frequency of T-bet + expressing CD4 + and CD8 + T cells by ~250% and ~650%, respectively (Fig. 5a, c, d, f).In comparison to BCG immunized mice, the frequency of T-bet + expressing CD4 + and CD8 + T-cells were increased by 130.0% and 285.0%, respectively, in ΔmenT4ΔT3 immunized mice, respectively (Fig. 5a, c, d, f).We also observed that the frequency of T-bet + expressing CD4 + and CD8 + T cells increased by 2.0-and 2.31-fold in BCG immunized mice as compared to naive mice (Fig. 5a, c, d, f).The frequency of IL-17A secreting CD4 + T cells in ΔmenT4ΔT3 immunized mice and BCG immunized mice increased by ~150% and 140%, respectively, in comparison to naive mice (Fig. S7a, b).This increase was observed to be non-significant in comparison to the proportion of cells obtained in naive mice.We also determined the levels of various cytokines in the supernatants of PPD-stimulated splenocytes.In agreement with our immunophenotyping experiments, the levels of IFN-γ were significantly increased in culture supernatants from PPDstimulated splenocytes obtained from BCG and ΔmenT4ΔT3 immunized groups in comparison to naive animals (Fig. 5g).Notably, TNF-α levels were increased by ∼1.6-fold and ~2.5-fold in culture supernatants from PPD-stimulated splenocytes obtained from the ΔmenT4ΔT3 immunized group in comparison to BCG immunized and naive group (Fig. 5h).The levels of IL-2 were also significantly increased in culture supernatants from PPD-stimulated splenocytes obtained from ΔmenT4ΔT3 immunized mice in comparison to naive animals (Fig. 5i).In agreement with FACS data, the levels of secreted IL-17A were not significantly changed in supernatants of PPD-stimulated splenocytes from naive, BCG and ΔmenT3ΔT4 immunized mice (Fig. 5j).Previously, it has been shown that the FoxP3 transcription factor is mostly expressed by regulatory T cells and dampens the antimicrobial inflammatory immune response 82,83 .Immunization of mice with ΔmenT4ΔT3 did not result in significant changes in the frequency of FoxP3 + CD4 + T-cells in comparison to naive mice (Fig. S7a, c).These observations suggest that ΔmenT4ΔT3 immunization-induced protection against M. tuberculosis challenge might be independent of Treg cells.Together, our data suggests that immunization with ΔmenT4ΔT3 results in the expansion of antigen-specific T H 1 response, which may contribute to protection against M. tuberculosis infection.
Furthermore, we also evaluated the effector and memory T cell response in spleens of immunized mice (Fig. S8).We observed a significant increase in the frequency of activated memory T helper and T cytotoxic cells by 2.70-fold and 3.80-fold, respectively, in ΔmenT4ΔT3 immunized mice and by ~1.60-and 2.30-fold, respectively, in mice immunized with BCG in comparison to naive mice (Fig. 6a-d).In comparison to BCG immunized mice, the frequency of activated memory CD4 + and CD8 + T cells were increased by ~1.70-and 1.60-fold, respectively, in ΔmenT4ΔT3 immunized mice (Fig. 6a-d).Furthermore, a slight increase, though not statistically significant, in the frequency of effector T helper cells was observed in ΔmenT4ΔT3 immunized as compared to naïve mice (from 14.96 to 17.62%) and BCG immunized mice (from 11.44 to 17.62%) (Fig. S9a, c).Moreover, in comparison to naive mice, the frequency of effector memory CD4 + T cells decreased from 18.2 to 13.26% in ΔmenT4ΔT3 immunized mice (Fig. S9a, d).We also observed that the frequency of effector memory CD4 + T cells was significantly reduced in ΔmenT4ΔT3 immunized mice in comparison to BCG immunized mice (Fig. S9a, d).However, the frequency of effector and effector memory CD8 + T cells were comparable in naïve and ΔmenT4ΔT3 immunized mice (Fig. S9b, f, g).In comparison to BCG immunized mice, we observed a significant reduction in the proportion of central memory CD4 + and CD8 + T cells in ΔmenT4ΔT3 immunized mice (Fig. S9a, b, e, h).The proportion of central memory CD4 + T cells was significantly reduced in ΔmenT4ΔT3 immunized mice relative to naive mice (Fig. S9a, e).The frequency of effector CD4 + T cells, effector memory CD4 + and CD8 + T cells and central memory CD4 + and CD8 + T cells were comparable in naive and BCG immunized mice (Fig. S9a-e, g, h).Taken together, we show that immunization of mice with ΔmenT4ΔT3 strain induces higher antigen-specific T H 1 response and expansion of activated memory T helper and cytotoxic T cell response in comparison to BCG immunized mice.These enhanced immune signatures might be associated with protection imparted by immunization with ΔmenT4ΔT3 against M. tuberculosis challenge.

Discussion
TA systems are mostly bicistronic genetic elements that are widely distributed in prokaryotes and are involved in stress adaptation, genome maintenance, pathogenesis and control of phage infection.M. tuberculosis genome encodes for >90 TA pairs, and most of these belong to either type II or MenAT TA systems 11,12,14 .TA systems belonging to type II subfamily have been extensively characterized in M. tuberculosis.However, very limited information is available about MenAT TA systems from M. tuberculosis.The three-dimensional structures of MenT1, MenT3 and MenT4 have been solved by X-ray crystallography, but their exact role in M. tuberculosis physiology and pathogenesis is still unknown [29][30][31] .Superimposition of these solved structures revealed that MenT toxins have conserved folds and catalytic sites 30,31 .In agreement with previous reports, we demonstrate that overexpression of MenT3 and MenT4 inhibits E. coli and M. tuberculosis growth 29,30 .As expected, co-expression of cognate antitoxins or mutation of amino acid residues in the highly conserved motifs abrogated the growth inhibition activity associated with these toxins.
Studies have shown that MenT1, MenT3 and MenT4 homologs from M. tuberculosis possess NTase activity in vitro and inhibit protein synthesis by preventing aminoacylation of tRNAs 30,31 .
Using temperature-sensitive mycobacteriophages, we generated M. tuberculosis mutant strains harboring deletions in either MenT3 or MenT4 or both MenT3 and MenT4.The growth patterns of single and double mutant strains were comparable to the parental strain.The relative abundance and upregulation of a subset of TA systems upon exposure to stress conditions and drugs suggests that these might function in a cumulative manner to enable M. tuberculosis to adapt to these conditions 12,21,22 .We observed that MenT3 and MenT4 are mutually redundant, but both MenT3 and MenT4 cumulatively contribute to the adaptation of M. tuberculosis upon exposure to oxidative stress.Complementation of the double mutant strain with menT3 partially restored the growth defect associated with the double mutant strain upon exposure to oxidative stress.We also observed that the relative transcript levels of menA3, menT3, menA4 and menT4 did not significantly change upon exposure to oxidative stress.It has been previously reported that in addition to their own promoter, antitoxins or TA complexes belonging to the type II subfamily can bind to other promoter sequences [84][85][86] .We hypothesize that by binding to the promoter of genes involved in oxidative stress adaptation, MenAT3 and MenAT4 might regulate their expression.We also demonstrate that the survival of parental, ΔmenT3, ΔmenT4 and ΔmenT4ΔT3 strains was similar after exposure to either nitrosative stress or nutritional stress or acidic stress.
Several studies have implicated the role of TA systems in the pathogenesis of various microorganisms.It has been shown that TA systems are essential for the virulence of bacterial pathogens such as H. influenzae, V. cholerae, S. typhimurium, S. aureus, and M. tuberculosis 10 .Here, we report that relative to the parental strain, ΔmenT4ΔT3 strain was attenuated for growth at both acute and chronic stages of infection in guinea pigs.As observed in the case of ΔmazF3Δ9Δ6 or ΔvapC22 or ΔhigB1 or ΔvapBC3 or ΔvapBC4 or ΔvapBC11 or ΔmenT2, the growth defect associated with ΔmenT4ΔT3 strain was more prominent in spleens and during the chronic stage of infection in guinea pigs 21,23,[25][26][27]35 . In greement with guinea pig data, we observed that the deletion of menT3 and menT4 also impaired the growth of M. tuberculosis in mice tissues.As expected, we observed significantly reduced tissue damage in lung sections from guinea pigs infected with the double mutant strain in comparison to parental strain infected animals.We also observed that complementation with either menT3 or menT4 did not restore the growth defect associated with double mutant strain in host tissues.These findings imply that MenT3 and MenT4 are mutually redundant and that simultaneous deletion of both toxins results in an attenuated phenotype in vivo.However, based on the available data, the possibility of acquisition of secondary site mutation during the generation of ΔmenT4ΔT3 can't be ruled out.In contrast to the in vivo phenotype, the growth patterns of both parental and ΔmenT4ΔT3 strains were comparable in macrophages.A possible reason for the lack of growth defect in macrophages could be that the function of MenT3 and MenT4 lies outside macrophages, and these proteins are involved in the interaction or intracellular growth of M. tuberculosis inside other host cells such as lung epithelial cells, dendritic cells, adipocytes, neutrophils and mesenchymal stem cells [87][88][89][90][91] .
Transcriptomics is widely used to gain a better understanding of the mechanisms for the attenuation of bacterial pathogens [92][93][94] .Bacterial RNA sequencing revealed that transcript levels of genes encoding for proteins involved in stress adaptation and virulence were reduced in mid-log phase cultures of the double mutant strain as compared to the parental strain.Therefore, we hypothesize that the in vivo attenuated phenotype of the mutant strain might be associated with the reduced expression of these proteins.In order to further unravel the plausible mechanisms associated with the in vivo growth defect of the mutant strain, we also compared the transcriptional profiles obtained from lung tissues of animals infected with either wild type or ΔmenT4ΔT3 strain.Detailed analysis of the RNA-seq data revealed that the levels of transcripts encoding for proteins involved in calcium signaling, apoptosis and autophagy were increased in ΔmenT4ΔT3 infected animals relative to parental strain infected animals (Fig. 7a).We also observed that the expression of proteins involved in inflammatory response was reduced in animals infected with the double mutant strain in comparison to parental strain infected animals (Fig. 7a).Taken together, the data suggests that the coordinated execution of these pathways might be associated with the observed growth defect of the mutant strain in host tissues.
Till date, BCG is the only licensed vaccine in use against TB.However, due to its limited efficacy, variable protection and adverse effects, there is an urgent need to develop new vaccine candidates that confer better protection against TB [95][96][97][98] .There have been several reports regarding the development of attenuated mutant strains and evaluating their ability to impart protection against M. tuberculosis in animal models.The ability of these strains to impart protection is most likely due to the fact that they harbor a complete repertoire of genes encoding for immunodominant antigens.Given that ΔmenT4ΔT3 was attenuated for growth in guinea pigs and Balb/c mice relative to the parental strain and was being further evaluated as a vaccine candidate, detailed immunological studies were performed in immunized C57BL/ 6 mice, a preferred model for immune studies.We observed that immunization of C57BL/6 mice with ΔmenT4ΔT3 significantly reduced the lung bacterial burdens in comparison to naive mice at both 4-and 10 weeks post-infection.The observed protection was similar to the levels seen in BCG-immunized mice.However, the reduction in splenic bacterial counts at 10 weeks post-infection was statistically significant in comparison to bacterial loads observed in BCG-immunized mice.In agreement with mice protection data, we observed that immunization with ΔmenT4ΔT3 was also able to impart protection against M. tuberculosis in guinea pigs.The protection imparted upon immunization with ΔmenT4ΔT3 at 4 weeks post-infection was significantly enhanced in comparison to protection observed in BCG-immunized guinea pigs.However, the levels of protection were comparable in ΔmenT4ΔT3 and BCG-immunized guinea pigs at 8 weeks postchallenge.Interestingly, despite similar bacterial loads in lung tissues, analysis of H&E-stained lung sections revealed larger alveolar spaces and minimal cellular infiltration in lung sections of guinea pigs immunized with ΔmenT4ΔT3 in comparison to BCG immunized animals.The levels of protection observed upon immunization of C57BL/ 6 and guinea pigs with ΔmenT4ΔT3 were similar to those reported for other attenuated vaccine candidates such as ΔleuD (in guinea pigs) or ΔpanCD (in guinea pigs) or ΔRD1ΔpanCD (in C57BL/6 mice) 73,99,100 .
Several studies have shown that antigen-specific T H 1 response is desired to impart protection against M. tuberculosis challenge in C57BL/6 mice and guinea pigs [76][77][78]81,101 . Previos studies have shown that protective immunity against TB depends on an acquired cellular immune response involving T-cell subsets, and a T H 1-type response is considered favorable in imparting protection against M. tuberculosis 102 .In agreement, we observed significant upregulation of IFN-γ and T-bet expression in CD4 + and CD8 + T cell compartments in ΔmenT4ΔT3 immunized mice in comparison to BCG-immunized mice.This T H 1 skewed response might be associated with the increased protection imparted by ΔmenT4ΔT3 against M. tuberculosis challenge in spleen tissues.Next in order to further understand the immune correlates of protection in the ΔmenT4ΔT3 immunized mice, we also evaluated the antigen-specific effector and memory T cell response in immunized mice.We observed increased activated memory T cell response in ΔmenT4ΔT3 immunized mice as compared to BCG immunized or naive mice.Since memory responses are desirable for vaccine-induced long-lasting protection, we speculate that increased activated memory T cell response, as observed in ΔmenT4ΔT3 immunized mice, might be able to impart protection against relapses as well.It is well documented that T H 1 immune responses are necessary for host defence against TB [103][104][105] .We also observed a significant increase in the secretion of T H 1 cytokines such as IFN-γ, TNF-α and IL-2 in culture supernatants of PPD stimulated splenocytes from ΔmenT4ΔT3 immunized mice, in agreement with the flow cytometry data.Studies have shown that IFN-γ and TNF-α are important for the effective control of M. tuberculosis infection 106 . IL- has also been shown to stimulate the growth of B-cells, T-cells and NK cells and is essential for cellular immunity and granuloma formation in M. tuberculosis infection 106 .We propose that increased amounts of these cytokines in culture supernatants from PPD stimulated splenocytes from ΔmenT4ΔT3 immunized mice might contribute to its ability to impart protection against M. tuberculosis challenge 81,[107][108][109][110] .However, limitations of the present study include lack of experiments to study immunological responses in guinea pigs, and a small number of animals were utilized in mice experiments.
Overall, this study reveals that although MenT3 and MenT4 are dispensable for in vitro growth, these toxins function in a cumulative manner and are essential for M. tuberculosis to establish disease in mice and guinea pigs.We also show that immunization with ΔmenT4ΔT3 is able to impart protection against M. tuberculosis in mice and guinea pigs.We propose that the protection observed in ΔmenT4ΔT3 immunized animals is most likely associated with increased antigen-specific T H 1 bIased and activated memory immune response (Fig. 7b).This study also provides the rationale for the development of live vaccines against TB based on the inactivation of virulence-associated pathways regulated by MenT3 and MenT4.Future studies include (1) unmarking and construction of ΔmenT4ΔT3-based multiple-allele mutant strains (such as panCD, leuD, metX, etc.), and these strains would be evaluated for safety and efficacy studies in compliance with the Geneva consensus 111 , (2) identification of small molecule inhibitors against MenT3 and MenT4 proteins and (3) experiments to validate RNA seq data obtained from infected lung tissues.

Ethics approval
Ethics approval for this work was obtained from the Institutional Biosafety Committee and Review Committee on Genetic Manipulation of the Department of Biotechnology, Ministry of Science and Technology, Government of India.

Bacterial strains, plasmids, and culture conditions
The list of strains and plasmids used in the study are listed in Supplementary Table 1.The list of primers used in the study are listed in Supplementary Table 2.For overexpression studies, genes encoding for MenT3, MenT4 and their point mutants were PCR amplified and cloned in either isopropyl thio-β-galactoside (IPTG)-or anhydrotetracycline (Atc) inducible vectors, pET28b or pTetR, respectively 112 .The recombinant constructs were verified by DNA sequencing.For coexpression studies, the toxin (menT3 or menT4) and antitoxin (menA2 or menA3 or menA4) were separately cloned into MCS-1 and MCS-2 of the pETDuet vector 113 .The bacterial strains were cultured in either LB broth or LB agar or 7H9 or 7H11 medium as previously described 114 .

Growth inhibition assays in E. coli and M. tuberculosis
In order to determine whether inducible expression of MenT3 and MenT4 results in growth inhibition of E. coli, BL-21 (λDE3, pLysS) was transformed with pET28b or pETDuet derivatives.The expression of proteins in recombinant E. coli strains was induced by the addition of 1.0 mM IPTG when OD 600nm of 0.4-0.6 was attained.The growth of recombinant strains was assayed by either measuring OD 600nm or by spotting diluted cultures on LB agar plates.For growth inhibition studies in M. tuberculosis, pTetR or pTetR-menT3 or pTetR-menT4 were electroporated in M. tuberculosis H 37 Rv.The expression of MenT3 and MenT4 in early-log phase cultures (OD 600 nm ~0.2) of recombinant strains was induced by the addition of 50 ng/ml Atc.The growth of parental and recombinant M. tuberculosis strains was determined by measuring OD 600nm and bacterial numbers at regular intervals.For CFU enumeration, 10.0-fold serial dilutions were prepared and plated on MB7H11 medium at 37 °C for 3-4 weeks.

Construction of various mutant and complemented strains of M. tuberculosis
The single and double mutant strains of M. tuberculosis Erdman were generated using temperature-sensitive mycobacteriophages 115 .Briefly, for the construction of ΔmenT4 strain, ~800 bp upstream and downstream region of menT4 was PCR amplified and cloned into cosmid vector, pYUB854 115 .The recombinant pYUB854-ΔmenT4 was Pac I digested and packaged into phagemid, pYUB159.The recombinant phagemid was electroporated in M. smegmatis to generate temperature-sensitive mycobacteriophages.These temperaturesensitive mycobacteriophages were used to transduce mid-log phase cultures of M. tuberculosis Erdman (OD 600nm ~0.8-1.0) to generate ΔmenT4 strain.For the generation of ΔmenT3 and ΔmenT4ΔT3 double mutant strain, ~800 bp upstream and downstream region of menT3 was PCR amplified and cloned into cosmid vector, pYUB854.The hygromycin resistance gene pYUB854-ΔmenT3 was replaced with the kanamycin resistance gene resulting in pYUB854-ΔmenT3-kan.The recombinant cosmid, pYUB854-ΔmenT3-kan, was packaged, and temperature-sensitive mycobacteriophages were prepared as described above.For the construction of ΔmenT3 and ΔmenT4ΔT3, mid-log phase cultures of M. tuberculosis Erdman or ΔmenT4 strain (OD 600nm ~0.8-1.0),respectively, were transduced with temperaturesensitive menT3 mycobacteriophages.The replacement of menT3 and menT4 with kanamycin and hygromycin resistance gene, respectively, in ΔmenT4ΔT3 strain of M. tuberculosis Erdman was verified by PCR and whole genome sequencing.In order to complement the ΔmenT4ΔT3 strain, menA3-menT3 and menA4-menT4 locus were PCR amplified along with 500 bp upstream region and cloned into pMV306apramycin.The recombinant construct was electroporated into ΔmenT4ΔT3 and transformants were selected on MB7H11 plates containing kanamycin, hygromycin and apramycin.

Stress experiments
For oxidative stress, early-log phase cultures of various strains (OD 600nm ~0.2) were exposed to 5 mM H 2 O 2 for either 24 or 72 h.For acidic or nitrosative stress, early log phase cultures were harvested by centrifugation, washed twice and resuspended in MB7H9 medium, pH-5.2 (acidic medium) for 7 days or in the presence of 5 mM NaNO 2 (nitrosative stress for 24 h).For nutritional stress, early-log phase cultures were harvested, washed twice and resuspended in 1x trisbuffered saline tween-80 for 7 days.At designated time points, CFU enumeration was performed by plating 10.0-fold serial dilutions on MB7H11 agar plates at 37 °C for 3-4 weeks.

Macrophage experiments
Human monocyte cell-line THP-1 was obtained from National Centre for Cell Science and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and differentiated into macrophages by overlaying cells with RPMI medium with 20 ng/ml of phorbol myristate acetate (PMA) for 24 h.For macrophage experiments, differentiated THP-1 cells (2 × 10 5 per well) were infected with various strains at a MOI of 1:10 for 4 h.Subsequently, macrophages were washed with 1x PBS, cultured in RPMI medium containing 200 µg/ml amikacin for 2 h to remove extracellular bacteria and overlaid with RPMI complete medium.The medium was replaced every 48 h during the experiment.At designated time points, macrophages were lysed using 1x PBS containing 0.1% triton X-100.The number of viable bacteria in lysates was determined by plating 10.0-fold serial dilutions on MB7H11 agar at 37 °C for 3-4 weeks.

Lipid extraction experiments
Briefly, various strains were grown to mid-log phase (OD 600nm ~0.8-1.0).The cultures were harvested by centrifugation and washed twice with 1x PBS.apolar and polar lipids were extracted as previously described 35 .An equal amount of different lipid fractions was loaded on silica plates and resolved using onedimensional TLC in the different solvent systems as described previously 35 .Different lipids were visualized by staining the TLC with 5% molybdophosphoric acid in ethanol and subsequent charring at 100 °C.TLC images were acquired using a gel documentation system (Bio-Rad).

Bacterial RNA sequencing
For bacterial RNA-seq experiments, total RNA was isolated from midlog phase cultures (OD 600nm ~0.8-1.0) of various strains using the Trizol method as previously described 114 .The quality of the isolated RNA was analyzed using Agilent Bioanalyzer.RNA samples were further processed for library preparation and sequenced using the Illumina HiSeq2000 platform.The sequenced reads were aligned to the M. tuberculosis Erdman sequence using the Hisat 2 program (Version 2.0.5).The differential gene expression analysis using aligned reads was performed using the Cuffdiff program of the Cufflinks package.

Animal husbandry
Six to eight weeks old outbred female Duncan Hartley guinea pigs (~250-300 g) were obtained from Disease Free Small Animal House, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar.Six to eight weeks old inbred female Balb/c and C57BL/6 mice (~20-25 g) were obtained from the Experimental Animal Facility, NCR Biotech Science Cluster, Faridabad.Animals were housed in a group of either 5 (mice) or 3 (guinea pigs) in individually ventilated cages in BSL-3 labs.The animals were maintained at a room temperature of 22 ± 3 °C, relative humidity of 30 to 70%, 15-20 air changes/h, light intensity of 325-350 lux with a 14 h light/10 h dark cycle and noise intensity of <85 db.A sterilized pellet diet (Altromin International, Germany) was offered ad libitum to the animals throughout the experimental period.Ad libitum aqua guard filtered, and autoclaved water was served in bottles fitted with stainless steel nozzles.

Animal virulence studies
The institutional animal ethics committee of the Translational Health Science and Technology Institute (THSTI) approved the animal experiments.The animal experiments were performed as per the guidelines provided by the committee for the control and supervision of experiments on animals.For aerosol infections, mid-log phase cultures (OD 600nm ~0.8-1.0) of various strains were harvested by centrifugation and washed twice with 1x PBS.Subsequently, single-cell suspensions were prepared, and cultures were diluted to 5 × 10 8 CFU (female Balb/c mice) or 1 × 10 8 CFU (female Duncan Hartley strain, guinea pigs) in 10 ml saline.The animals were infected using a Glass-col aerosol generation device that implanted ~100 bacilli in lung tissues.For CFU analysis, lungs and spleens were homogenized in saline, 10.0fold serial dilutions were prepared and plated on MB7H11 agar for 3-4 weeks at 37 °C.For histopathology analysis, lung tissues were fixed in 10% formalin, embedded in paraffin wax and stained with hematoxylin and eosin (H&E).The stained sections were analyzed for tissue damage by a histopathologist.The total granuloma score in H&Estained lung sections from infected guinea pigs was determined as described earlier 116 .

Host RNA-seq analysis
For host RNA-seq analysis, total RNA was isolated from lung tissues of uninfected Balb/c mice and those infected with either wild type or ΔmenT4ΔT3 at 4 weeks post-infection using Qiagen RNA extraction protocol as per manufacturer recommendations 26 .RNA was subjected to DNase I treatment, and the quality and integrity of RNA were analyzed using Agilent Bioanalyzer and subjected to sequencing.The paired-end reads obtained were analyzed for quality control using the NGS QC Toolkit, and those with a Phred score of >Q30 were carried forward for analysis.HISAT2 splice-aware read aligner was used for the alignment of the passed reads to the reference Mus musculus (mm10) genome.Stringtie was employed for the assembly and quantification of the aligned reads of all samples.Analysis for differential expression was performed using DESeq2.The heat maps were prepared using TB tools.

Protective efficacy studies
For immunization experiments, female mice (C57BL/6) or female guinea pigs (Duncan Hartley strain) were immunized with either saline or single-cell suspension containing 5 × 10 5 CFU of BCG or ΔmenT4ΔT3 strain via subcutaneous route in mice or 1 × 10 5 CFU via intradermal route in guinea pigs.For protection studies, at 10 weeks post-immunization, animals were challenged with M. tuberculosis H 37 Rv using a Glass Col aerosol chamber as described above.The protective efficacy imparted by immunization with either BCG or ΔmenT4Δ3 was determined by bacterial enumeration and histopathological analysis as described above.

Flow cytometry experiments
At 6 weeks post-immunization spleens from naive and immunized C57BL/6 mice were isolated and passed through a 70 μm cell strainer to obtain a single-cell suspension.Subsequently, RBCs were lysed with ice-cold ammonium-chloride-potassium (ACK) lysis buffer (20-30 s at room temperature) and resuspended in RPMI complete media.For immunophenotyping experiments, splenocytes were seeded in a 96-

Fig. 1 |
Fig. 1 | Functional characterization of MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems from M. tuberculosis.a-f Overexpression of MenT3 and MenT4 inhibits the growth of E. coli and M. tuberculosis.a, b These panels show growth patterns of E. coli Bl-21 (pLysS, λDE3) strains harboring pET28b derivatives expressing either wild type or mutant MenT3 (a) or wild type or mutant MenT4 (b) proteins in the absence or presence of inducer.c, d These panels depict growth patterns of E. coli BL21 (pLysS, λDE3) strains harboring pET-Duet constructs overexpressing MenT3 (c) or MenT4 (d) either alone or along with their cognate or non-cognate antitoxins.The growth of various strains was determined by measuring OD 600nm .The data shown in these panels are representative of two independent experiments.e, f The growth patterns of M. tuberculosis H 37 Rv harboring pTetR derivatives expressing either MenT3 or MenT4 are shown in these panels.The growth of recombinant strains was determined by measuring either OD 600nm (e) or bacterial counts (f).The data shown in (e) is representative of two independent experiments.The data shown in (f) is mean ± SD of log 10 CFU obtained from two independent experiments, each performed with duplicate cultures.p values depicted on the graphs were assessed using one-way ANOVA.g, h Construction of ΔmenT4ΔT3 strain of M. tuberculosis.g Schematic representation of menT3 and menT4 locus in parental, ΔmenT4 and ΔmenT4ΔT3 strain of M. tuberculosis Erdman is shown.The open reading frame of menT4 was replaced with the hygromycin resistance gene in ΔmenT4 strain of M. tuberculosis.In the double mutant strain,ΔmenT4ΔT3, the open reading frame of menT3 and menT4 were replaced with kanamycin and hygromycin resistance gene, respectively.h The replacement of menT3 and menT4 with kanamycin and hygromycin resistance gene, respectively, in their respective single and double mutant strain was confirmed by PCR using genespecific primers.Source Data are provided as a Source Data file.

Fig. 2 |
Fig. 2 | MenT3 and MenT4 are cumulatively essential for M. tuberculosis to survive in oxidative stress and establish infection in guinea pigs.The growth of wild type and ΔmenT4ΔT3 was compared after exposure to either oxidative (a) or nitrosative (b) or nutritional (c) or acidic (d) stress.The data shown in these panels are mean ± SD of log 10 CFU obtained from two (a, c, d) or three (b) independent experiments, each performed with duplicate cultures.e THP-1 macrophages were infected with various strains, and the number of intracellular bacteria was determined at different time points.The data shown in this panel are mean ± SD of log 10 CFU obtained from triplicate wells and representative of two independent experiments performed in duplicates or triplicates.The data shown in these panels in mean ± SD of log 10 CFU in lungs (f) and spleens (g) of guinea pigs (Duncan Hartley strain) infected with either wild type or ΔmenT4ΔT3 strain at 4-and 8-weeks postinfection.The data shown in these panels in mean ± SD of log 10 CFU obtained from 6 animals (except in (f) week 8, wild type n = 4 and in (g) week 4, ΔmenT4ΔT3 n = 5 and week 8, wild type n = 5).The data shown for the 4-week time point are representative of two independent experiments.The data shown for the 8-week time point are obtained from a single experiment.h The data shown in this panel are mean ± SD of total granuloma score in H&E-stained sections of guinea pigs infected with wild type or ΔmenT4ΔT3 strain of M. tuberculosis at 4-and 8-weeks postinfection.The data shown are obtained from 6 animals from a single experiment (except in week 8, wild type n = 4).i This panel shows representative images of H&Estained sections of lung tissues of guinea pigs infected with either wild type or ΔmenT4ΔT3 strain at 4-or 8-week post-infection.Scale bar, 100 μm.p values depicted on the graphs were assessed using a two-tailed paired t-test.Source Data are provided as a Source Data file.

Fig. 3 |
Fig. 3 | Global transcriptional changes in lung tissues of mice infected with either wild type or ΔmenT4ΔT3 strain at 4 weeks post-infection.a, b Deletion of menT3 and menT4 attenuates M. tuberculosis growth in lungs and spleens of mice.The data shown in these panels are mean ± SD of log 10 CFU in lungs (a) or spleens (b) of infected Balb/c mice at 4-and 8-weeks post-infection obtained from 5 animals.The data shown in these panels for 4-week time point are representative of two independent experiments.The data shown for the 8-week time point are obtained from a single experiment.p values depicted on the graphs were assessed using a two-tailed paired t-test.c-g Host transcriptional profiles of lung tissues from uninfected or mice infected with either wild type or ΔmenT4ΔT3 strain.c Venn diagram depicting correlation of expression profiles obtained from lung tissues of either uninfected or mice infected with parental or ΔmenT4ΔT3 strain at 4 weeks post-infection.d Volcano plot comparing transcription profiles obtained from lung tissues of mice infected with either wild type or ΔmenT4ΔT3 strain at 4 weeks postinfection.The transcripts with increased or decreased expression have been shown as blue or red dots, respectively.The black dots represent the transcripts that remain unchanged and are not statistically different between these two groups.e-g Heat maps showing transcripts with differential expression in mice infected with either wild type or ΔmenT4ΔT3 strain at 4 weeks post-infection.The transcripts with differential expression in mice infected with these strains are involved in either calcium signaling (e) or apoptosis/autophagy (f), or immune response (g).The color intensity in heatmaps represents the log 2 value of normalized expression counts.The data shown in (c-g) are obtained from three independent biological replicates.Source Data are provided as a Source Data file.

Fig. 4 |
Fig. 4 | Immunization of mice and guinea pigs with ΔmenT4ΔT3 imparts protection against M. tuberculosis challenge.a 6-8 weeks old female C57BL/6 mice were immunized subcutaneously with 5 × 10 5 CFU of either M. bovis BCG or ΔmenT4ΔT3.At 10 weeks post-immunization, animals were challenged with M. tuberculosis and bacterial enumeration was performed at 4-and 10 weeks postinfection.b-e The data shown in these panels is mean ± SD of log 10 CFU in lungs (b, d) and spleens (c, e) of naive or BCG or ΔmenT4ΔT3 immunized mice after challenge with M. tuberculosis at 4 weeks (b, c) and 10 weeks (d, e) post-infection.The data shown in these panels are mean ± SD of log 10 CFU obtained from 5 animals from a single experiment (except in (e) week 10, ΔmenT4ΔT3 n = 4).f 6-8-week-old female guinea pigs (Duncan Hartley strain) were immunized intradermally with 1 × 10 5 CFU of either M. bovis BCG or ΔmenT4ΔT3.At 10 weeks post-immunization, animals were challenged with M. tuberculosis and bacterial enumeration and histopathology analysis were performed at 4 and 8-weeks post-infection.The bacterial burdens in the lungs of naive or BCG or ΔmenT4ΔT3 immunized guinea pigs were determined after challenge with M. tuberculosis at 4 (g) and 8 weeks (h) postchallenge.The data shown are mean ± SD of log 10 CFU obtained from 6 animals from a single experiment (except in (g) week 4, naïve and BCG n = 7 and in (h) week 8, BCG n = 8).The data shown in this panel are mean ± SD of total granuloma score obtained from H&E stained lung sections of naive or BCG or ΔmenT4ΔT3 immunized guinea pigs at 4 weeks (i) and 8 weeks (j) post-challenge.The data shown are obtained from 6 animals from a single experiment (except in (i) week 4, naïve and BCG n = 7 and in (j) week 8, BCG n = 8).k This panel shows representative images of H&E-stained sections of lung tissues of naive or immunized guinea pigs after infection with M. tuberculosis for 4 weeks or 8 weeks.Scale bar, 100 μm.p values depicted on the graphs were assessed using one-way ANOVA.Source Data are provided as a Source Data file.

Fig. 5 |
Fig. 5 | Immunization of mice with ΔmenT4ΔT3 results in increased antigenspecific T H 1-response. a-f C57BL/6 mice immunized with saline or BCG or ΔmenT4ΔT3 were sacrificed at 6 weeks post-immunization, and intracellular cytokines were measured in PPD-stimulated splenocytes.a Representative FACS plots depicting percentage frequency of CD4 + IFN-γ + and CD4 + T-bet + T cells in spleens of naive or BCG or ΔmenT4ΔT3 immunized mice.These panels show mean ± SD of percentage frequency CD4 + IFN-γ + (b) and CD4 + T-bet + (c) in spleens of naive or BCG or ΔmenT4ΔT3 immunized mice.d Representative FACS plots depicting percentage frequency of CD8 + IFN-γ + and CD8 + T-bet + T cells in spleens of naive or BCG or ΔmenT4ΔT3 immunized mice.These panels show the percentage frequency of CD8 + IFN-γ + (e) and CD8 + T-bet + (f) T cells in the spleens of naive or BCG or ΔmenT4ΔT3 immunized mice.The data shown in (b, c, e, f) are mean ± S.D. of the proportion of T-cells obtained from five animals from a single experiment.The levels of secreted IFN-γ (g), TNF-α (h), IL-2 (i), IL-17A (j) were measured in the culture supernatants from PPD-stimulated splenocytes by ELISA.The data shown are mean ± SD of cytokine levels in PPD-stimulated splenocytes obtained from five animals from a single experiment (except in (h), BCG n = 4).p values depicted on the graphs were assessed using one-way ANOVA.Source Data are provided as a Source Data file.

Fig. 6 |
Fig. 6 | Immunization of mice with ΔmenT4ΔT3 increases the expansion of activated memory T cell response.Representative FACS plots showing percentage frequency of CD4 + activated memory T H cells (CD4 + CD44 + CD69 + , a) and CD8 + activated memory T C cells (CD8 + CD44 + CD69 + , b) in spleens of naive or BCG or ΔmenT4ΔT3 immunized C57BL/6 mice.These panels show the percentage frequency of activated memory CD4 + T H cells (c) and CD8 + T C cells (d) in spleens of naive or BCG or ΔmenT4ΔT3 immunized mice.The data shown in these panels are mean ± SD of the percentage frequency of cells obtained from five animals from a single experiment.p values depicted on the graphs were assessed using one-way ANOVA.Source Data are provided as a Source Data file.

Fig. 7 |
Fig. 7 | Proposed model for attenuation of ΔmenT4ΔT3 in host tissues and for protection imparted by ΔmenT4ΔT3 against M. tuberculosis.a In the present study, we show that deletion of menT3 and menT4 impairs the growth of M. tuberculosis in mice and guinea pigs.The attenuated phenotype of ΔmenT4ΔT3 is most likely associated with increased levels of transcripts encoding for proteins involved in calcium signaling, autophagy, apoptosis and activation of macrophages and neutrophils.The increased expression of these pathways results in the generation of a robust innate immune response that restricts M. tuberculosis growth and disease progression.b Here, we show that administration of ΔmenT4ΔT3 imparts protection in mice and guinea pigs against challenge with M. tuberculosis.The immunization of mice with ΔmenT4ΔT3 strain results in the expansion of activated memory immune response and induction of antigen-specific T H 1 in comparison to naive and BCG immunized mice.This significant increase in the generation of activated memory and T H 1 immune response might be associated with protection against M. tuberculosis challenge.The figure has been prepared using BioRender.

Table 1 |
Differential expression of genes in ΔmenT4ΔT3 strain relative to the wild type strain